Light-activated cell identification and sorting (LACIS) for selection of edited clones on a nanofluidic device.

ABSTRACT: Despite improvements in the CRISPR molecular toolbox, identifying and purifying properly edited clones remains slow, laborious, and low-yield. Here, we establish a method to enable clonal isolation, selection, and expansion of properly edited cells, using OptoElectroPositioning technology for single-cell manipulation on a nanofluidic device. Briefly, after electroporation of primary T cells with CXCR4-targeting Cas9 ribonucleoproteins, single T cells are isolated on a chip and expanded into colonies. Phenotypic consequences of editing are rapidly assessed on-chip with cell-surface staining for CXCR4. Furthermore, individual colonies are identified based on their specific genotype. Each colony is split and sequentially exported for on-target sequencing and further off-chip clonal exp

SUBMITTER: Mocciaro A 

PROVIDER: S-EPMC6123811 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature